Approximately one in 5000 males in human population suffers from coagulation disorder, hemophilia A. This disease is primarily caused by deficiency in the factor VIII gene located in the X-chromosome and is difficult to treat by conventional medicine. Current treatment of hemophilia A by intravenous infusion of factor VIII concentrates is very costly and has a potential side effect of developing inhibitors. Gene therapy, on the other hand, can potentially prevent these limitations of current treatments. Although recombinant adeno-associated virus (rAAV) vectors are promising for deliver factor VIII gene, applying AAV vector technology to Hemophilia A lagged behind other genetic diseases because of this size constraint (limited to ~5kb). To improve factor VIII gene delivery utilizing rAAV vectors, we have developed novel engineered factor VIII molecules for use in both dual vectors and single vector strategy. The specific aims for this proposal are: 1). To develop a Factor VIII heavy chain molecule efficient for secretion;2). To develop and characterize a novel mini factor VIII gene for rAAV delivery;3). To analyze immune responses against the engineered factor VIII transgene products. The success of this proposal may lead to a clinical trial of hemophilia A using AAV vectors.